Methods for fractional quatification of cholesterol in lipoproteins and quantification reagents

ABSTRACT

The present invention provides a method for the quantitative determination of cholesterol in low density lipoproteins and a reagent kit for use therein. The present invention also provides a method for continuous fractional determination of cholesterol in high density lipoproteins and cholesterol in low density lipoproteins and a reagent kit for use therein, as well as a method for continuous fractional determination of cholesterol in high density lipoproteins and total cholesterol and a reagent kit for use therein.

This application is a National Stage of International Application No.PCT/JP99/04128 filed Jul. 30, 1999.

TECHNICAL FIELD

The present invention relates to a method for the quantitativedetermination of cholesterol in low density lipoproteins (LDL)(hereinafter referred to as LDL cholesterol), which is important in thefield of clinical diagnosis, and a reagent for use in the method. Thepresent invention also relates to a method for the continuous fractionaldetermination of cholesterol in high density lipoproteins (HDL)(hereinafter referred to as HDL cholesterol) and LDL cholesterol, whichare also important in the field of clinical diagnosis, and a reagent kitfor use therein. The present invention further relates to a method forthe continuous fractional determination of HDL cholesterol, LDLcholesterol and total cholesterol [the term is used to mean totalcholesterol in HDL, LDL, very low density lipoproteins (hereinafterreferred to as VLDL) and chylomicron (hereinafter referred to as CM)],which are important in the field of clinical diagnosis, as well as areagent kit to be used therefor.

BACKGROUND ART

In general, HDL is called good cholesterol since HDL functions to removecholesterol accumulated on arterial walls and transport cholesterol toliver. On the other hand, LDL is generally termed bad cholesterolbecause of its action to transport cholesterol to peripheral tissuesincluding arterial walls. In the field of clinical investigations, thelevels of HDL cholesterol, LDL cholesterol and total cholesterol areuseful indices for total judgement of lipid-related diseases such asarteriosclerosis, etc.

These cholesterol levels are separately determined using reagentsexclusively specific to each type of cholesterol so that an autoanalyzeris designed so as to be suitable for individual determination of thesecholesterol levels. It has been desired to further improve thespecificity of a reagent to each cholesterol. Besides, no simple andautomated method for the continuous fractional determination of HDLcholesterol, LDL cholesterol, total cholesterol, etc in the samedetection system is known.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a method for thequantitative determination of LDL cholesterol and a determinationreagent for use in such a method.

Another object of the present invention is to provide a method for thecontinuous fractional determination of HDL cholesterol and LDLcholesterol in the same sample and a reagent kit for use therein.

More specifically, the present invention relates to (1) through (27)below.

(1) A method for quantitatively determining LDL cholesterol in abiological sample, which comprises

performing the reaction of cholesterol in the presence of:

a) a biological sample,

b) cholesterol esterase and cholesterol oxidase or cholesteroldehydrogenase (hereinafter collectively referred to as CH enzymes), and

c) a reagent enabling the CH enzymes of b) to act only on LDLcholesterol, and

measuring the amount of the hydrogen peroxide or reduced coenzyme formedby the reaction to quantitatively determine the concentration of LDLcholesterol.

(2) The method according to (1), wherein the reagent enabling CH enzymesto act only on LDL cholesterol is a reagent containing at least apolyoxyethylene derivative and a polyoxyethylene-polyoxypropylenecopolymer.

(3) The method according to (2), wherein the polyoxyethylene derivativeis a polyoxyethylene alkyl ether or a polyoxyethylene alkylaryl ether.

(4) The method according to (2) or (3), wherein thepolyoxyethylene-polyoxypropylene copolymer is a surfactant representedby general formula (I):

HO—(C₂H₄O)_(a)—(C₃H₆O)_(b)—(C₂H₄O)_(c)—H  (I)

(wherein a, b and c, which may be the same or different, each representsan integer of 1 to 200).

(5) A method for the continuous fractional determination of HDLcholesterol and LDL cholesterol in a biological sample, which comprises

subjecting cholesterol to the first reaction in the presence of:

a) a biological sample,

b) CH enzymes, and

c) a reagent enabling the CH enzymes of b) to act only on HDLcholesterol, and

measuring the amount of the hydrogen peroxide or reduced coenzyme formedby the first reaction to quantitatively determine the concentration ofHDL cholesterol,

then adding

d) a reagent enabling the CH enzymes of b) to act only on LDLcholesterol,

subjecting cholesterol to the second reaction, and

measuring the amount of the hydrogen peroxide or reduced coenzyme formedby the second reaction to quantitatively determine the concentration ofLDL cholesterol.

(6) A method for the continuous fractional determination of HDLcholesterol and LDL cholesterol in a biological sample, which comprises

subjecting cholesterol to the first reaction in the presence of:

a) a biological sample,

b) CH enzymes, and

c) a reagent enabling the CH enzymes of b) to act only on HDLcholesterol, and

measuring the amount of the hydrogen peroxide or reduced coenzyme formedby the first reaction to quantitatively determine the concentration ofHDL cholesterol,

then adding

d) CH enzymes, and

e) a reagent enabling the CH enzymes of d) to act only on LDLcholesterol,

subjecting cholesterol to the second reaction, and

measuring the amount of the hydrogen peroxide or reduced coenzyme formedby the second reaction to quantitatively determine the concentration ofLDL cholesterol.

(7) The method according to (5) or (6), wherein the reagent enabling CHenzymes to act only on LDL cholesterol is a reagent containing at leasta polyoxyethylene derivative and a polyoxyethylene-polyoxypropylenecopolymer.

(8) The method according to (7), wherein the polyoxyethylene derivativeis a polyoxyethylene alkyl ether or a polyoxyethylene alkylaryl ether.

(9) The method according to (7) or (8), wherein thepolyoxyethylene-polyoxypropylene copolymer is a surfactant representedby general formula (I):

HO—(C₂H₄O)_(a)—(C₃H₆O)_(b)—(C₂H₄O)_(c)—H  (I)

(wherein a, b and c, which may be the same or different, each representsan integer of 1 to 200).

(10) A method for the continuous fractional determination of HDLcholesterol and total cholesterol in a biological sample, whichcomprises

subjecting cholesterol to the first reaction in the presence of:

a) a biological sample,

b) CH enzymes, and

c) a reagent enabling CH enzymes of b) to act only on HDL cholesterol,and

measuring the amount of the hydrogen peroxide or reduced coenzyme formedby the first reaction to quantitatively determine the concentration ofHDL cholesterol,

then adding

d) a reagent enabling the CH enzymes of b) to act on cholesterol in alllipoproteins,

subjecting cholesterol to the second reaction, and

measuring the amount of the hydrogen peroxide or reduced coenzyme formedby the second reaction to quantitatively determine the concentration oftotal cholesterol.

(11) A method for the continuous fractional determination of HDLcholesterol and total cholesterol in a biological sample, whichcomprises

subjecting cholesterol to the first reaction in the presence of:

a) a biological sample,

b) CH enzymes, and

c) a reagent enabling the CH enzymes of b) to act only on HDLcholesterol, and

measuring the amount of the hydrogen peroxide or reduced coenzyme formedby the first reaction to quantitatively determine the concentration ofHDL cholesterol,

then adding

d) CH enzymes, and

e) a reagent enabling the CH enzymes of d) to act on cholesterol in alllipoproteins,

subjecting cholesterol to the second reaction, and

measuring the amount of the hydrogen peroxide or reduced coenzyme formedby the second reaction to quantitatively determine the totalcholesterol.

(12) The method according to any one of (5) through (11), wherein thereagent enabling CH enzymes to act only on cholesterol in HDL is areagent for aggregating lipoproteins other than HDL.

(13) The method according to (12), wherein the reagent for aggregatinglipoproteins other than HDL further contains a nonionic surfactant thatdoes not solubilize the aggregated lipoproteins.

(14) The method according to (12) or (13), wherein the reagent foraggregating lipoproteins other than HDL is a reagent comprising heparinor a salt thereof, phosphotungstic acid or a salt thereof, dextransulfuric acid or a salt thereof, polyethylene glycol, sulfonatedcyclodextrin or a salt thereof, sulfonated oligosaccharide or a saltthereof, or a mixture thereof and a divalent metal salt.

(15) The method according to (6) or (11), wherein the CH enzymes used inthe first reaction of cholesterol are chemically modified enzymes andthe CH enzymes used in the second reaction of cholesterol are enzymesthat are not chemically modified.

(16) The method according to any one of (10) through (15), wherein thereagent enabling the CH enzymes to act on cholesterol in alllipoproteins is a reagent containing a lipoprotein solubilizingsurfactant.

(17) A reagent for determining LDL cholesterol comprising CH enzymes anda reagent enabling the CH enzymes to act only on LDL cholesterol.

(18) The reagent for determining LDL cholesterol according to (17),wherein the reagent enabling the CH enzymes to act only on LDLcholesterol is a reagent containing at least a polyoxyethylenederivative and a polyoxyethylene-polyoxypropylene copolymer.

(19) The reagent according to (18), wherein the polyoxyethylenederivative is a polyoxyethylene alkylaryl ether.

(20) The reagent according to (18) or (19), wherein thepolyoxyethylene-polyoxypropylene copolymer is a surfactant representedby general formula (I):

HO—(C₂H₄O)_(a)—(C₃H₆O)_(b)—(C₂H₄O)_(c)—H  (I)

(wherein a, b and c, which may be the same or different, each representsan integer of 1 to 200).

(21) A reagent kit for the fractional determination of HDL cholesteroland LDL cholesterol comprising a first reagent and a second reagent,said first reagent comprising a reagent for aggregating lipoproteinsother than HDL lipoprotein and a reagent containing CH enzymes, and saidsecond reagent comprising a reagent enabling CH enzymes to act only onLDL cholesterol.

(22) The reagent kit according to (21), wherein the reagent enabling CHenzymes to act only on LDL cholesterol is a reagent containing apolyoxyethylene derivative and a polyoxyethylene-polyoxypropylenecopolymer.

(23) The reagent kit according to (21), wherein the polyoxyethylenederivative is a polyoxyethylene alkylaryl ether.

(24) The reagent kit according to (21) or (22), wherein thepolyoxyethylene-polyoxypropylene copolymer is a surfactant representedby general formula (I):

HO—(C₂H₄O)_(a)—(C₃H₆O)_(b)—(C₂H₄O)_(c)—H  (I)

(wherein a, b and c, which may be the same or different, each representsan integer of 1 to 200).

(25) A reagent kit for the fractional determination of HDL cholesteroland total cholesterol comprising a first reagent and a second reagent,said first reagent comprising a reagent for aggregating lipoproteinsother than HDL lipoprotein and a reagent containing CH enzymes, and saidsecond reagent comprising a reagent enabling CH enzymes to act oncholesterol in all lipoproteins.

(26) The reagent kit according to (25), wherein the reagent enabling CHenzymes to act on cholesterol in all lipoproteins further contains alipoprotein solubilizing surfactant.

(27) The reagent kit according to any one of (21) through (26), whereinthe reagent for aggregating lipoproteins other than HDL lipoprotein is areagent comprising heparin or a salt thereof, phosphotungstic acid or asalt thereof, dextran sulfuric acid or a salt thereof, polyethyleneglycol, sulfonated cyclodextrin or a salt thereof, sulfonatedoligosaccharide or a salt thereof, or a mixture thereof and a divalentmetal salt.

Hereinafter the present invention will be described in more detail.

As described above, the present invention relates to a method for thequantitative determination of LDL cholesterol which comprises adding toa biological sample containing various types of lipoproteins a specificreagent enabling CH enzymes to act only on LDL cholesterol (hereinafterreferred to as reagent A) and a reagent for use in such a method.

As described above, the present invention also relates to a method forthe fractional determination of HDL cholesterol and LDL cholesterolwhich comprises adding to a biological sample containing various typesof lipoproteins a specific reagent enabling CH enzymes to act only onHDL cholesterol (hereinafter referred to as reagent B) to quantitativelydetermine HDL cholesterol and then adding reagent A to quantitativelydetermine LDL cholesterol as well as a reagent kit for use therein.

As described above, the present invention further relates to a methodfor the fractional determination of HDL cholesterol and totalcholesterol which comprises adding to a biological sample containingvarious types of lipoproteins the reagent B to quantitatively determineHDL cholesterol and then adding to the reaction mixture a reagentenabling CH enzymes to act on cholesterol in all lipoproteins(hereinafter referred to as reagent C) to quantitatively determine totalcholesterol as well as a reagent kit for use therein.

A biological sample to which the present invention is to be applied isnot particularly limited. More specifically, blood itself or bloodfractions such as plasma or serum, etc. may be used as the sample.

The reactions for quantitatively determining cholesterol in the presentinvention are generally carried out in an aqueous medium, preferably ina buffer solution.

Buffers useful in the buffer solution includetris(hydroxymethyl)aminomethane, phosphate buffer, borate buffer andGood's buffer. Examples of Good's buffer areN-(2-acetamido)-2-aminoethanesulfonic acid (ACES),N-(2-acetamido)iminodiacetic acid (ADA), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO),N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES),2-(N-morpholino)ethanesulfonci acid (hereinafter referred to as MES),3-(N-morpholino)propanesulfonic acid (hereinafter referred to as MOPS),3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO),piperazine-N,N′-bis(2-ethanesulfonic acid) (hereinafter referred to asPIPES), piperazine-N,N′-bis(2-hydroxypropane-3-sulfonic acid) (POPSO),etc.

The pH of the buffer solution is 5 to 10, preferably 6 to 9. Theconcentration of the buffer to be used is 5 to 500 mM, preferably 20 to200 mM.

The reagent A which enables CH enzymes to act only on LDL cholesterol isa reagent that does not enable CH enzymes to act on cholesterol in HDL,VLDL and CM. The reagent A also enables CH enzymes to act only on LDLcholesterol even in the presence of a reagent for aggregatinglipoproteins other than HDL, which will be later described.

The reagent A is typically a reagent containing at least apolyoxyethylene derivative and a polyoxyethylene-polyoxypropylenecopolymer.

Suitable polyoxyethylene derivative is exemplified by a polyoxyethylenealkylaryl ether, a polyoxyethylene alkyl ether, etc., having the alkylmoiety of at least 8 carbon atoms, e.g., octyl, nonyl, etc. and havingthe aryl moiety being phenyl, etc.

Specific examples of the polyoxyethylene derivative include commerciallyavailable Nonion HS-210, Nonion HS-215, Nonion NS-208.5 and NonionHS-208 (all produced by NOF Corporation) and Emulgen L-40, Emulgen 911and Emulgen 810 (all produced by Kao Corporation). Thehydrophile-lipophile balance (hereinafter referred to as HLB) of thepolyoxyethylene derivative is preferably 9 to 20.

The polyoxyethylene-polyoxypropylene copolymers may be either randomcopolymers or block copolymers of polyoxyethylene and polyoxypropylene.An example of the copolymer is a compound represented by general formula(I):

HO—(C₂H₄O)_(a)—(C₃H₆O)_(b)—(C₂H₄O)_(c)—H  (I)

(wherein a, b and c, which may be the same or different, each representsan integer of 1 to 200).

Examples of the compounds represented by general formula (I) includecommercially available compounds such as Pluronic L-121, Pluronic L-122,Pluronic L-101, Pluronic P-103 and Pluronic F-108 (all produced by AsahiDenka Kogyo K. K.). The molecular weight of the polypropylene glycolmoiety in the compounds of general formula (I) is preferably at least2,050, more preferably 2,750 or more, most preferably 3,250 or more. TheHLB of the polyoxyethylene-polyoxypropylene copolymers is in preferably1 to 6.

The respective concentration of the polyoxyethylene derivatives andpolyoxyethylene-polyoxypropylene copolymers used is not specificallylimited but is preferably 0.001 to 10%, more preferably 0.01 to 5%, mostpreferably 0.05 to 1%.

Examples of the reagent B that enables CH enzymes to act only on HDLcholesterol are reagents for aggregating lipoproteins other than HDL andantibodies to lipoproteins other than HDL.

Reagents for aggregating lipoproteins other than HDL are generally thosecontaining agents for aggregating these lipoproteins and/or divalentmetal salts. Examples of the aggregating agent include heparin or saltsthereof, phosphotungstic acid or salts thereof, dextran sulfuric acid orsalts thereof, polyethylene glycol, sulfated cyclodextrin or saltsthereof, sulfated oligosaccharide or salts thereof, and mixturesthereof. Examples of the cyclodextrin include α-cyclodextrin,β-cyclodextrin and γ-cyclodextrin. Examples of the oligosaccharideinclude maltotriose, maltotetraose, maltopentaose, maltohexaose andmaltoheptaose. Examples of the salts include sodium, potassium, lithium,ammonium and magnesium salts. Examples of the divalent metal saltinclude magnesium, calcium, manganese and nickel salts.

Preferable examples of the aggregating agent used include 0.02 to 10 mMheparin having a molecular weight of 5, 000 to 20, 000 or salts thereof,0.1 to 10 mM phosphotungstic acid having a molecular weight of 4,000 to8,000 or salts thereof, 0.01 to 5 mM dextran sulfuric acid having amolecular weight of 10,000 to 500,000 or salts thereof, 0.1 to 20 mMdextran sulfuric acid having a molecular weight of 1,000 to 10,000 orsalts thereof, 0.3 to 100 mM polyethylene glycol (PEG) having amolecular weight of 4,000 to 25,000, 0.1 to 50 mM sulfated cyclodextrinhaving a molecular weight of 1,000 to 3,000 or salts thereof, 0.1 to 50mM sulfated oligosaccharide having a molecular weight of 400 to 3,000 orsalts thereof, and mixtures thereof. More preferred examples are 0.03 to1 mM heparin having a molecular weight of 14,000 to 16,000 or saltsthereof, 0.1 to 3 mM phosphotungstic acid having a molecular weight of5,000 to 7,000 or salts thereof, 0.01 to 5 mM dextran sulfuric acidhaving a molecular weight of 150,000 to 250,000 or salts thereof, 0.1 to10 mM dextran sulfuric acid having a molecular weight of 1,000 to 5,000or salts thereof, 1.0 to 50 mM PEG having a molecular weight of 5,000 to22,000, 0.1 to 10 mM sulfated cyclodextrin having a molecular weight of1,000 to 2,000 or salts thereof, 0.1 to 10 mM sulfated oligosaccharidehaving a molecular weight of 400 to 2,000 or salts thereof, and mixturesthereof.

Preferred examples of the divalent metal salt include the salts ofmagnesium, calcium, manganese, nickel and cobalt, the concentration ofwhich is 0.1 to 50 mM. Preferably, the magnesium salt is used in aconcentration of 0.1 to 50 mM.

It is preferred that the agents for aggregating lipoproteins other thanHDL further contain a nonionic surfactant that does not dissolve theaggregated lipoproteins.

Examples of the nonionic surfactant that does not dissolve theaggregated lipoproteins include a polyoxyethylene alkyl ether, apolyoxyethylene alkylaryl ether, a polyoxyethylene-polyoxypropylenecopolymer, a polyoxyethylene alkyl ether sulfuric acid salts and analkylbenzene sulfonate. Among these surfactants, polyoxyethylene ethers[Emulgen 220 (Kao Corporation), etc.] are particularly desired as thepolyoxyethylene alkyl ether; commercially available Emulgen 66, etc. asthe polyoxyethylene alkyl aryl ether; commercially available PluronicF88 (Asahi Denka Kogyo K. K.) as the polyoxyethylene-polyoxypropylenecondensate, commercially available Emal 20C (Kao Corporation) as thepolyoxyethylene alkyl ether sodium sulfate, and sodium dodecylbenzenesulfonate as the alkyl benzenesulfonic acid salt.

The nonionic surfactant that does not dissolve the aggregatedlipoproteins can be used in combination, so long as the surfactant doesnot enable CH enzymes to act on LDL cholesterol. However, it ispreferable to use the nonionic surfactant solely. The concentration ofthe nonionic surfactant is not particularly limited but is preferably0.01 to 10%, more preferably 0.1 to 5%.

Examples of the antibodies to lipoproteins other than HDL include anantiapo-lipoprotein B antibody, an antiapo-lipoprotein C antibody, anantiapo-lipoprotein E antibody and an anti-β-lipoprotein antibody. Theseantibodies may be employed solely or in combination. The antibodies maybe either polyclonal or monoclonal. The antibodies may also bechemically or enzymatically degraded or modified.

As the reagent C enabling CH enzymes to act on cholesterol in alllipoproteins, there are, for example, surfactants that dissolve alllipoproteins.

As the surfactants above, there are used nonionic surfactants thatdissolve HDL, LDL, VLDL and CM. Specific examples of such surfactantsare nonionic surfactants commercially available as Triton X-100,polyoxyethylene alkyl ethers such as Emulgen 106, Emulgen 108, Emulgen709, etc. These surfactants may be used solely or in combination. Theconcentration of the surfactants is not particularly limited but ispreferably 0.01 to 10%, more preferably 0.1 to 5%.

As the enzymes having the activities of cholesterol esterase,cholesterol oxidase and cholesterol dehydrogenase which may be used inthe present invention, there are, for example, cholesterol esterase andlipoprotein lipase derived from microorganisms or animals having theability to hydrolyze cholesterol ester, cholesterol oxidase derived frommicroorganisms having the ability to oxidize cholesterol to producehydrogen peroxide, and cholesterol dehydrogenase derived frommicroorganisms or animals.

These enzymes can be employed depending upon specificity to substrate.In the case of the quantitative determination of HDL cholesterol, it ispreferred to use an enzyme specific to the cholesterol and for thequantitative determination of LDL cholesterol, an enzyme specificthereto is preferably used. In order to further improve the specificityand stability of these enzymes, enzymes that are chemically modifiedwith a group having polyethylene glycol as a main component, awater-soluble oligosaccharide residue, or a sulfopropyl group may alsobe used. Furthermore, enzymes obtained by genetic engineering may alsobe used.

Examples of the reagent for modifying the enzymes (chemical modifier)include compounds wherein polyethylene glycol and a group capable ofbonding to an amino group are connected, e.g. Sun Bright VFM4101 (NOFCorporation) wherein polyethylene glycol and a group capable of bondingto an amino group such as N-hydroxysuccinimido group are connected, SunBright AKM series, ADM series and ACM series [all manufactured by NOFCorporation, Chemical Engineering Monographs (Kagaku Kogaku Ronbunshu),20 (3), 459 (1994)] which are compounds having the polyalkylene glycolstructure and the acid anhydride structure, compounds wherein apolyethylene glycol-polypropylene glycol copolymer and a group capableof bonding to an amino group are connected, copolymers of polyethyleneglycol monomethacryl monomethyl ether and maleic anhydride, etc.Furthermore, activated polyurethane P4000 (Boehringer Mannheim,Directions for Enzyme Modification Set) which is a polyurethane chemicalmodifier, Dextran T40, which is a dextran chemical modifier, andactivated TCT (Boehringer Mannheim, Directions for Enzyme ModificationSet), 1,3-propanesultone, etc. may also be used. By the use of thesechemical modifiers, the enzymes can be modified with a group havingpolyethylene glycol as a main component, a group having polypropyleneglycol as a main component, a group having a copolymer of polypropyleneglycol and polyethylene glycol, a group containing a saccharide in thestructure, a sulfopropyl group, a polyurethane group, etc.

A method for the reaction of an enzyme with the above chemical modifieris described in Yuji Inada, “Tanpakushitu-no-Hybrid (Hybrid ofProteins)” published by Kyoritsu Publishing Co. (1987), etc. Typically,when using, e.g., Sun Bright, the enzyme is dissolved in a buffersolution such as HEPES buffer of pH 8 or above, then, e.g.,0.01-500-fold molar amount of Sun Bright is added to the solution at 0°C. to 50° C., followed by stirring for 5 to 60 minutes. The resultingreaction mixture is used as it is, or if necessary, after removal of lowmolecular weight compounds with ultrafilter.

According to the present invention, the cholesterol esterase,cholesterol oxidase and cholesterol dehydrogenase are preferably used inthe reaction mixture at a concentration of 0.01 to 200 U/ml, morepreferably 0.1-100 U/ml.

In the present invention, the CH enzymes used to quantitativelydetermine HDL cholesterol may be used as they are for quantitativedetermination of LDL cholesterol or cholesterol other than HDL or totalcholesterol.

Alternatively, for the quantitative determination of LDL cholesterol orcholesterol other than HDL or total cholesterol, CH enzymes having thesame or different specificities may be newly added to the system.

Preferably, chemically modified CH enzymes are used for the quantitativedetermination of HDL cholesterol and for the determination ofcholesterol in lipoproteins other than LDL or HDL or total cholesterol,CH enzymes without any chemical modification are used.

In the reaction of cholesterol according to the present invention, asurfactant or cholic acid which is conventionally used to activate CHenzymes may also be employed as far as they do not affect the reactionspecificity. Further, various salts for solubilizing proteins such asglobulin may also be used.

As the surfactant for activating the CH enzymes, anionic surfactants areused, e.g., at a concentration of 0 to 1%. Examples of the cholic acidare cholic acid, deoxycholic acid, taurocholic acid and chenodeoxycholicacid. The cholic acid is used at a concentration of 0 to 5%. Examples ofthe anionic surfactant include an alkyl sulfonate such as1-pentasulfonate, 1-hexasulfonate, 1-heptasulfonate and1-octasulfonate.These surfactants are used at a concentration of 0 to 5%.

Examples of the salts include sodium chloride, sodium sulfate, potassiumchloride, potassium sulfate, magnesium chloride, magnesium sulfate,magnesium acetate, lithium chloride, lithium sulfate, ammonium chloride,ammonium sulfate, magnesium nitrate and calcium nitrate. These salts areused at a concentration of 0 to 100 mM.

When the reaction of cholesterol is carried out with cholesterolesterase and cholesterol oxidase, hydrogen peroxide is formed. Theformed hydrogen peroxide can be quantitatively determined, using e.g.4-aminoantipyrine and a phenol, 4-aminoantipyrine and Trinder's reagent,or a highly sensitive chromogen in the presence of peroxidase.

Examples of phenols are phenol, 4-chlorophenol, m-cresol and3-hydroxy-2,4,6-triiodobenzoic acid (HTIB).

Examples of the Trinder's reagents (General Catalog of Dojin KagakuKenkyusho, 19th ed., 1994) are anilines such as N-sulfopropylaniline,N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (TOOS),N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS),N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS),N-ethyl-N-sulfopropyl-m-toluidine (TOPS),N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS),N,N-dimethyl-m-toluidine, N,N-disulfopropyl-3,5-dimethoxyaniline,N-ethyl-N-sulfopropyl-m-anisidine, N-ethyl-N-sulfopropylaniline,N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline,N-sulfopropyl-3,5-dimethoxyaniline,N-ethyl-N-sulfopropyl-3,5-dimethylaniline,N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-anisidine,N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline andN-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline,N-ethyl-N-(3-methylphenyl)-N′-succinylethylenediamine (EMSE), andN-ethyl-N-(3-methylphenyl)-N′-acetylethylenediamine.

As the highly sensitive chromogen, there are10-(N-methylcarbamoyl)-3,7-bis(dimethylamino)phenothiadine (MCDP)disclosed in Japanese Published Examined Patent Application No.33479/85, bis[3-bis(4-chlorophenyl)methyl-4-dimethylaminophenyl]amine(BCMA) disclosed in Japanese Published Examined Patent Application No.27839/92, the compounds disclosed in Japanese Published UnexaminedPatent Application No. 296/87, etc.

The chromogen is preferably used in a concentration of 0.01 to 10 mg/ml.

When the reaction of cholesterol is carried out with cholesterolesterase and cholesterol dehydrogenase in the presence of an oxidizedcoenzyme, NAD(P), as a substrate, a reduced coenzyme, NAD(P)H, isformed. The formed NAD(P)H can be quantitatively determined by measuringthe absorbance of a reaction mixture at 300 to 500 nm, preferably 330 to400 nm, particularly preferably about 340 nm. The determination ofNAD(P)H may otherwise be made by forming a formazan pigment throughaddition of diaphorase and a tetrazolium salt and then measuring theformazan pigment by colorimetry.

The reaction for the quantitative determination of LDL cholesterol iscarried out at 10 to 50° C., preferably 30 to 40° C., usually at 37° C.,for 1 to 30 minutes, preferably 2 to 10 minutes.

In the fractional determination, the reaction for quantitativelydetermining HDL cholesterol (hereinafter referred to as the firstreaction) are carried out at 10 to 50° C., preferably 30 to 40° C.,usually at 37° C., for 1 to 30 minutes, preferably 2 to 10 minutes; thereactions for quantitatively determining LDL cholesterol or totalcholesterol (hereinafter referred to as the second reaction) are carriedout at 10 to 50° C., preferably 30 to 40° C., usually at 37° C., for 1to 30 minutes, preferably 2 to 10 minutes. The start of the secondreaction may be at any stage, e.g., after the first reaction issubstantially completed or during the first reaction, so long as thequantitative determination of HDL is completed. The second reaction isinitiated by adding the reagent A enabling the CH enzymes to actspecifically on LDL cholesterol or the reagent C enabling the CH enzymesto act on cholesterol in all lipoproteins and, if necessary, CH enzymes.The hydrogen peroxide or reduced coenzyme [NAD(P)H] thus formed by thesecond reaction is quantitatively determined using the same reagents asused in the first reaction as they are, or, if necessary and desired,reagents may be newly added to the system.

In the present invention where HDL cholesterol and LDL cholesterol arefractionally determined by first performing the reaction of HDLcholesterol followed by the reaction of LDL cholesterol, the reaction ofLDL cholesterol is initiated by adding the reagent A as described above.In this case, when the first reaction of HDL cholesterol is carried outby adding the nonionic surfactant that does not dissolve the aggregatedlipoproteins other than HDL, i.e., by adding either the polyoxyethylenederivative or the polyoxyethylene-polyoxypropylene copolymer, the secondreaction of LDL cholesterol may also be initiated by adding such asurfactant as forming the reagent A in combination with the surfactantused in the reaction of HDL cholesterol.

The concentration of cholesterol in each lipoprotein is calculated bythe following equation based on a difference in absorbance (ΔOD) beforeand after each reaction using a test sample and a difference inabsorbance (ΔODstd) using a sample with a known concentration ofcholesterol in various lipoproteins.

The concentration of LDL cholesterol can be determined by the followingequation:

ΔOD÷ΔODstd×(known concentration of LDL cholesterol)

The concentration of HDL cholesterol can be determined by, e.g., thefollowing equation:

ΔOD÷ΔODstd×(known concentration of HDL cholesterol)

In the fractional determination, when the compounds formed in the firstand second reactions are the same and they are detected by the samemethod, the concentration of total cholesterol can be calculatedaccording to the following equation, using the difference in absorbancebefore the first reaction and after the second reaction:

ΔOD÷ΔODstd×(known concentration of total cholesterol)

The reagent of the present invention for quantitatively determining LDLcholesterol comprises CH enzymes and a reagent comprising thepolyoxyethylene derivative and the polyoxyethylene-polyoxypropylenecopolymer. The above reagent for quantitatively determining LDLcholesterol may further contain, if necessary, the aforesaid buffers,reagents for aggregating lipoproteins other than HDL, surfactants usedfor quantitatively determining cholesterol, cholic acids, various salts,enzymes such as peroxidase, chromogens such as 4-aminoantipyrine andTrinder's reagents or oxidized coenzymes such as NAD(P).

The reagent kit of the present invention for the fractionaldetermination of HDL cholesterol and LDL cholesterol comprises a firstreagent and a second reagent. For example, the first reagent comprises areagent containing an aggregating agent for lipoproteins other than HDLand CH enzymes and the second reagent comprises a reagent containing thepolyoxyethylene derivative and the polyoxyethylene-polyoxypropylenecopolymer.

The reagent kit of the present invention for the fractionaldetermination of HDL cholesterol and total cholesterol comprises a firstreagent and a second reagent. For example, the first reagent comprises areagent containing an aggregating agent for lipoproteins other than HDLand CH enzymes and the second reagent comprises a reagent containing anonionic surfactant that dissolves all lipoproteins (HDL, LDL, VLDL andCM).

The first and second reagents of the reagent kit in accordance with thepresent invention may further contain, if necessary and desired, theaforesaid buffers, surfactants used for the quantitative determinationof cholesterol, cholic acids, various salts, enzymes such as peroxidase,chromogens such as 4-aminoantipyrine and Trinder's reagents, oxidizedcoenzymes such as NAD(P).

In the second reagent, the source of CH enzymes may be the same as ordifferent from the first reagent. It is preferred that the chemicallymodified enzyme described above is used as the CH enzyme for the firstreagent and a CH enzyme not chemically modified is used as the CH enzymefor the second reagent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the correlation between the concentration ofHDL cholesterol obtained by the method of the present invention(designated by DB HDL-C in the figure) and the concentration of HDLcholesterol obtained by the comparative method (L HDL-C method,designated by S HDL-C in the figure).

FIG. 2 is a graph showing the correlation between the concentration ofLDL cholesterol obtained by the method of the present invention(designated by DB LDL-C in the figure) and the concentration of LDLcholesterol obtained by the comparative method (L LDL-C method,designated by S LDL-C in the figure).

FIG. 3 is a graph showing the correlation between the concentration oftotal cholesterol obtained by the method of the present invention(designated by DB-TC in the figure) and the concentration of totalcholesterol obtained by the comparative method (Determiner L TC IImethod, designated by L TC II in the figure).

FIG. 4 is a graph showing the correlation between the concentration ofHDL cholesterol obtained by the method of the present invention(designated by DB HDL-C in the figure) and the concentration of HDLcholesterol obtained by the comparative method (L HDL-C method,designated by S HDL-C in the figure).

FIG. 5 is a graph showing the correlation between the concentration ofLDL cholesterol obtained by the method of the invention (designated byMethod of the invention in the figure) and the concentration of LDLcholesterol obtained by the comparative method (designated byComparative method in the figure) which is calculated in accordance withthe Friedewald formula of conversion.

BEST MODES FOR CARRYING OUT THE INVENTION EXAMPLE 1

Determination of HDL Cholesterol and LDL Cholesterol

First reagent (pH = 7) MES (Nacalai Tesque, Inc.) 20 mM Dextran sulfonicacid (Tokyo Kasei) 0.23 mg/ml Magnesium sulfate (Kanto Chemical 1.5mg/ml Co., Ltd.) HDAOS (Dojin Kagaku) 0.23 mg/ml 4-Aminoantipyrine(Saikyo Kasei) 0.13 mg/ml Polyethylene glycol-modified 0.25 U/mlcholesterol esterase(*1) Polyethylene glycol-modified 1.65 U/mlcholesterol oxidase(*2) Peroxidase(Toyobo Co., Ltd.) 12.5 U/ml Secondreagent (pH = 7) MES (Nacalai Tesque, Inc.) 20 mM Cholesterol esterase(lipoprotein 3 U/ml lipase, Toyobo Co., Ltd.) Cholesterol oxidase (KyowaHakko 2 U/ml Kogyo Co., Ltd.) Pulronic L-121 (Asahi Denka Kogyo K.K.)0.7% Emulgen L-40 (Kao Corporation) 0.5% Calcium chloride (Wako PureChemical 0.1 mg/ml Industries, Ltd.) (*1): Prepared by dissolving 50 gof cholesterol esterase (Amano Pharmaceutical Co. Ltd.) in 1 L of 0.1 MHEPES buffer (pH 8.5) and adding 330 g of Sun Bright VFM4101 to thesolution at 25° C., followed by stirring for 2 hours. (*2): Prepared bydissolving 50 g of cholesterol oxidase (Kyowa Hakko Kogyo Co., Ltd.) in1 L of 0.1 M HEPES buffer (pH 8.0) and adding 10 g of Sun Bright VFM4101to the solution at 15° C., followed by stirring for 2 hours.

Serum samples from 30 healthy subjects were prepared and HDL cholesteroland LDL cholesterol in the samples were determined by the followingprocedures.

Reagent 1, 2.25 ml, was mixed with 30 μl of the sample. After themixture was stirred, absorbance E1 was immediately measured at 585 nm.The mixture was then incubated at 37° C. for 5 minutes, and absorbanceE2 at the same wavelength was measured. To the reaction solution wasadded 0.75 ml of Reagent 2. After the mixture was stirred, absorbance E3was immediately measured at 585 nm and after the mixture was incubatedat 37° C. for 5 minutes, absorbance E4 was measured at the samewavelength. Sera having known concentration of cholesterol were treatedby substantially the same procedure to measure absorbances E1std, E2std,E3std and E4std, respectively.

The concentration of HDL cholesterol was determined by the followingequation, using the absorbance data.

(E2−E1)÷(E2std−E1std)×(known concentration of HDL cholesterol)

The concentration of LDL cholesterol was likewise determined by thefollowing equation, using the absorbance data.

(E4−E3)÷(E4std−E3std)×(known concentration of LDL cholesterol)

For comparison, the concentration of HDL cholesterol and LD cholesterolin each serum sample was determined using Determiner L HDL-C andDeterminer L LDL-C (both manufactured by Kyowa Medex Co., Ltd.), whichare commercial kits for independent determination of the cholesterol,respectively. The coefficient of correlation between the resultsobtained with these commercial kits and the results according to themethod of the present invention was calculated. The coefficient ofcorrelation showed 0.997 for the HDL cholesterol and 0.988 for the LDLcholesterol.

FIG. 1 shows a correlation between the concentration (mg/dL) of HDLcholesterol according to the method of this invention (designated as DBHDL-C in FIG. 1) and the concentration (mg/dL) of HDL cholesterolobtained by the comparative method (L HDL-C method, designated as SHDL-C in FIG. 1). FIG. 2 shows a correlation between the concentration(mg/dL) of LDL cholesterol according to the method of this invention(designated as DB LDL-C in FIG. 2) and the concentration (mg/dL) of LDLcholesterol obtained by the comparative method (L LDL-C method,designated as S LDL-C in FIG. 2).

EXAMPLE 2

Determination of HDL Cholesterol and LDL Cholesterol

First reagent (pH = 7) MES (Nacalai Tesque, Ltd.) 20 mM Phosphotungsticacid (Wako Pure 7.5 mg/ml Chemical Industries, Ltd.) Magnesium sulfate(Wako Pure Chemical 1.5 mg/ml Industries, Ltd.) TOOS (Dojin Kagaku) 0.5mg/ml Emulgen B66 (Kao Corporation) 10 mg/ml 4-Aminoantipyrine (SaikyoKagaku) 0.5 mg/ml Cholesterol esterase (LPBP, 4 U/ml Asahi ChemicalIndustry Co., Ltd.) Cholesterol oxidase 2 U/ml (rCO, Oriental Yeast Co.,Ltd.) Peroxidase (Toyobo Co., Ltd.) 10 U/ml Second reagent (pH = 7) MES(Nacalai Tesque, Inc.) 50 mM Cholesterol esterase (lipoprotein 3 U/mllipase, Toyobo Co., Ltd.) Cholesterol oxidase (Kyowa Hakko 2 U/ml KogyoCo., Ltd.) Pulronic L-121 (Asahi Denka Kogyo K.K.) 0.7% Emulgen L-40(Kao Corporation) 0.5% Calcium chloride (Wako Pure Chemical 0.1 mg/mlIndustries, Ltd.)

To determine HDL cholesterol and LDL cholesterol, substantially the sameprocedure as in Example 1 was repeated using the same samples as inExample 1 except that the wavelength measured was changed to 555 nm. Thecoefficient of correlation between the results obtained with thecommercial kits of Determiner L HDL-C and Determiner L HDL-C and theresults obtained according to the method of the present invention wascalculated. The coefficient of correlation showed 0.929 for the HDLcholesterol and 0.911 for the LDL cholesterol.

EXAMPLE 3

Determination of HDL Cholesterol and Total cholesterol

First reagent (pH = 7) MES (Nacalai Tesque, Inc.) 20 mM Dextran sulfonicacid (Tokyo Kasei) 0.23 mg/ml Magnesium sulfate (Kanto Chemical 1.5mg/ml Co., Ltd.) HDAOS (Dojin Kagaku) 0.23 mg/ml 4-Aminoantipyrine(Saikyo Kasei) 0.13 mg/ml Polyethylene glycol-modified 0.25 U/mlcholesterol esterase (*1) Polyethylene glycol-modified cholesteroloxidase (*1) 1.65 U/ml Peroxidase 12.5 U/ml Second reagent (pH = 6.75)MES (Nacalai Tesque, Inc.) 30 mM Triton X-100 (Sigma) 1 g/L Cholesterolesterase (Toyobo Co., Ltd.) 2.4 U/ml Cholesterol oxidase (Amano 6.25U/ml Pharmaceutical Co., Ltd.) (*1): Prepared by the same procedure as*1 in Example 1 (*2): Prepared by the same procedure as *2 in Example 1

Serum samples from 30 healthy subjects used in Example 1 were preparedand HDL cholesterol and LDL cholesterol of the samples were determinedby the following procedures.

Reagent 1, 2.25 ml, was mixed with 30 μl of the sample. After themixture was stirred, absorbance E1 was immediately measured at 585 nm.The mixture was then incubated at 37° C. for 5 minutes, and absorbanceE2 was measured at the same wavelength. To the reaction solution wasfurther added 0.75 ml of Reagent 2. After the mixture was stirred,absorbance E3 was immediately measured at 585 nm and after the mixturewas incubated at 37° C. for 5 minutes, absorbance E4 was measured at thesame wavelength. Separately, sera having known concentration ofcholesterol were treated by substantially the same procedure to measureabsorbances E1std, E2std, E3std and E4std, respectively.

The concentration of HDL cholesterol was determined by the followingequation, using the absorbance data.

(E2−E1)÷(E2std−E1std)×(known concentration of HDL cholesterol)

The concentration of the total cholesterol was also determined by thefollowing equation, using the absorbance data.

(E4−E1)÷(E4std−E1std)×(known concentration of the total cholesterol)

For comparison, the concentration of HDL cholesterol and the totalcholesterol in each serum sample was determined using Determiner L HDL-Cand Determiner L TC II (both manufactured by Kyowa Medex Co., Ltd.),which are commercial kits for independent determination of thecholesterol, respectively. The coefficient of correlation between theresults obtained with the commercial kits and the results according tothe method of the present invention was calculated. The coefficient ofcorrelation showed 0.992 for the HDL cholesterol and 0.999 for the totalcholesterol.

FIG. 3 shows a correlation between the concentration (mg/dL) of thetotal cholesterol according to the method of this invention (designatedas DB-TC in FIG. 3) and the concentration (mg/dL) of the totalcholesterol obtained by the comparative method (Determiner L TC IImethod, designated as L TC II in FIG. 3).

FIG. 4 shows a correlation between the concentration (mg/dL) of HDLcholesterol according to the method of this invention (designated as DBHDL-C in FIG. 4) and the concentration (mg/dL) of HDL cholesterolobtained by the comparative method (L HDL-C method, designated as SHDL-C in FIG. 4).

EXAMPLE 4

First reagent (pH 7.25) PIPES (Nacalai Tesque, Inc.) 50 mM HDAOS (DojinKagaku) 0.3 mg/mL Second reagent (pH 7.25) PIPES (Nacalai Tesque, Inc.)50 mM Cholesterol esterase (lipoprotein lipase, 5 U/mL Toyobo Co., Ltd.)Cholesterol oxidase (Kyowa Hakko 1 U/mL Kogyo Co., Ltd.) Peroxidase(Toyobo Co., Ltd.) 20 U/mL 4-Aminoantipyrine (Saikyo Kasei) 0.51 mg/mLCalcium chloride (Wako Pure Chemical 0.1 mg/mL Industries, Ltd.)Surfactant (kind and concentration given in Table 1)

As samples, HDL, LDL, VLDL and CM separated from human blood serum bythe ultracentrifugation method were used. The respective lipoproteinfractions were provided by Fukushi Iryo Gijutsu Shinkoukai (WelfareMedical Technology Promotion Organization). These fractions wereprepared in accordance with Adv. Lipid Res., 6 (1968) [Practical methodsfor plasma lipoprotein analysis by Hatch, F. & Lees, R.]. Theconcentration of cholesterol in each lipoprotein used in this test wasdetermined using Determiner L TC II (Kyowa Medex Co., Ltd.). Theconcentration was found to be 73 mg/dL for HDL, 264 mg/dL for LDL, 84mg/dL for VLDL and 17 mg/dL for CM.

After 4 μL of each sample was mixed with 300 μL of the first reagent,the mixture was maintained at 37° C. for 5 minutes. At this stage, anabsorbance of the mixture was measured. Thereafter, 100 μL of the secondreagent was added to the mixture and reacted. After 5 minutes, anabsorbance of the reaction mixture was measured. The absorbance wasmeasured at a principal wavelength of 600 nm and a secondary wavelengthof 700 nm, using Hitachi 7070 autoanalyzer.

Differences in absorbance obtained before and after the reactions usingLDL fraction, HDL fraction, VLDL fraction and CM fraction are shown byA_(LDL), A_(HDL), A_(VLDL) and A_(CM), respectively.

The results are shown in Table 1 in terms of A_(HDL)/A_(LDL),A_(VLDL)/A_(LDL) and A_(CM)/A_(LDL), respectively. The results mean thatas the ratio becomes smaller, the conditions for quantitativedetermination are more specific to LDL.

TABLE 1 Concentra- A_(HDL)/ A_(VLDL)/ Surfactant tion (%) A_(LDL)A_(LDL) A_(CM)/A_(LDL) Pluronic L-121 0.2 7.3 6.6 4.6 Emulgen L40 0.16Pluronic L-121 0.2 9.6 13.5 3.2 Nonion HS-210 0.1 Pluronic L-121 0.210.2 7.7 1.2 Emulgen 911 0.1 Pluronic L-122 0.2 8.1 8.2 3.4 Emulgen L400.16 Pluronic L-121 0.2 34.7 47.9 16.8 (comparative example 1) EmulgenL-40 0.16 27.8 39.7 9.7 (comparative example 2) Nonion HS-210 0.1 35.535.5 6.1 (comparative example 3) Nonion HS-215 0.16 76.8 33.6 4.7(comparative example 4) Nonion NS-208.5 0.24 44.5 32.4 51.2 (comparativeexample 5) Nonion NS-208 0.08 30.2 47.3 28.3 (comparative example 6)Emulgen 911 0.1 22.6 15.9 3.0 (comparative example 7) Emulgen 810 0.224.7 36.8 5.8 (comparative example 8) Pluronic L-122 0.2 38.1 64.1 19.0(comparative example 9)

As shown in Table 1, the results reveal that by using the surfactants incombination, the reaction of cholesterol is more specific to LDLcholesterol than the case of using the surfactant alone.

EXAMPLE 5

First reagent (pH 6.75) MOPS (Nacalai Tesque, Inc.) 50 mM HDAOS (DojinKagaku) 0.3 mg/mL Second reagent (pH 6.75) MOPS (Nacalai Tesque, Inc.)50 mM Cholesterol esterase (lipoprotein lipase, 1 U/mL Toyobo Co., Ltd.)Cholesterol oxidase (Kyowa Hakko 3 U/mL Kogyo Co., Ltd.) Peroxidase(Toyobo Co., Ltd.) 20 U/mL 4-Aminoantipyrine (Saikyo Kasei) 0.51 mg/mLCalcium chloride (Wako Pure Chemical 0.1 mg/mL Industries, Ltd.)Surfactant (kind and concentration given in Table 2)

The test was carried out in a manner similar to Example 4 except forusing the surfactants shown in Table 2. A_(LDL), A_(HDL), A_(VLDL) andA_(CM) were thus determined, respectively, based on which the ratios ofA_(HDL)/A_(LDL), A_(VLDL)/A_(LDL) and A_(CM)/A_(LDL) were calculated.The concentration of cholesterol in each lipoprotein used in this testwas determined using Determiner L TC II (Kyowa Medex Co., Ltd.) and wasfound to be 81 mg/dL for HDL, 263 mg/dL for LDL, 72 mg/dL for VLDL and14 mg/dL for CM.

The results are shown in Table 2.

TABLE 2 Concentra- A_(HDL)/ A_(VLDL)/ Surfactant tion (%) A_(LDL)A_(LDL) A_(CM)/A_(LDL) Pluronic L-101 0.2 8.7 7.3 2.6 Emulgen L-40 0.16Pluronic P-103 0.2 13.0 3.9 1.7 Emulgen L-40 0.16 Pluronic F-108 0.215.0 4.5 1.4 Emulgen L-40 0.16 Emulgen L-40 0.16 26.5 24.6 4.7(comparative example 10) Pluronic L-101 0.2 19.0 14.3 5.6 (comparativeexample 11) Pluronic P-103 0.2 24.8 3.5 1.1 (comparative example 12)Pluronic F-108 0.2 28.8 17.8 1.6 (comparative example 13)

As shown in Table 2, the results reveal that by using the surfactants incombination, the reaction of cholesterol is more specific to LDLcholesterol than the case of using the surfactant alone.

EXAMPLE 6

First reagent (pH 6.75) MOPS (Nacalai Tesque, Inc.) 20 mM HDAOS (DojinKagaku) 0.3 mg/mL Second reagent (pH 6.75) MOPS (Nacalai Tesque, Inc.)20 mM Cholesterol esterase (lipoprotein lipase, 2 U/mL Toyobo Co., Ltd.)Cholesterol oxidase (Kyowa Hakko 3 U/mL Kogyo Co., Ltd.) Peroxidase(Toyobo Co., Ltd.) 20 U/mL 4-Aminoantipyrine (Saikyo Kasei) 0.51 mg/mLCalcium chloride (Wako Pure Chemical 0.1 mg/mL Industries, Ltd.)Surfactant (kind and concentration given in Table 3)

The test was carried out in a manner similar to Example 4 except forusing the surfactants shown in Table 3. A_(LDL), A_(HDL), A_(VLDL) andA_(CM) were determined, respectively, based on which the ratios ofA_(HDL)/A_(LDL), A_(VLDL)/A_(LDL) and A_(CM)/A_(LDL) were calculated.The concentration of cholesterol in each lipoprotein used in this testwas determined with Determiner L TC II (manufactured by Kyowa Medex Co.,Ltd.) and was found to be 85 mg/dL for HDL, 252 mg/dL for LDL, 75 mg/dLfor VLDL and 19 mg/dL for CM.

The results are shown in Table 3.

TABLE 3 Concen- Surfactant tration (%) A_(HDL)/A_(LDL) A_(VLDL)/A_(LDL)A_(CM)/A_(LDL) Pluronic L-121 0.7 4.0 5.0 3.4 Emulgen L-40 0.5

As shown in Table 3, LDL cholesterol can be more specifically determinedby using the combination of surfactants.

EXAMPLE 7

First reagent (pH 7.0) MOPS (Nacalai Tesque, Inc.) 10 mM HDAOS (DojinKagaku) 0.3 mg/mL Second reagent (pH 7.0) MOPS (Nacalai Tesque, Inc.) 50mM Cholesterol esterase (lipoprotein lipase, 1 U/mL Toyobo Co., Ltd.)Cholesterol oxidase (Kyowa Hakko Kogyo Co., Ltd.) 3 U/mL Peroxidase(Toyobo Co., Ltd.) 20 U/mL 4-Aminoantipyrine (Saikyo Kasei) 0.51 mg/mLCalcium chloride (Wako Pure Chemical 0.1 mg/mL Industries, Ltd.)Surfactant (kind and concentration given in Table 4)

The test was carried out in a manner similar to Example 4 except forusing the surfactants shown in Table 4. A_(LDL), A_(HDL), A_(VLDL) andA_(CM) were determined, respectively, based on which the ratios ofA_(HDL)/A_(LDL), A_(VLDL)/A_(LDL) and A_(CM)/A_(LDL) were calculated.The concentration of cholesterol in each lipoprotein used in this testwas determined using Determiner L TC II (Kyowa Medex Co., Ltd.) and wasfound to be 79 mg/dL for HDL, 273 mg/dL for LDL, 76 mg/dL for VLDL and16 mg/dL for CM.

The results are shown in Table 4.

TABLE 4 Concen- Surfactant tration (%) A_(HDL)/A_(LDL) A_(VLDL)/A_(LDL)A_(CM)/A_(LDL) Pluronic L-121 0.4 2.5 5.8 1.3 Emulgen L-40 0.32

As shown in Table 4, LDL cholesterol can be determined more specificallyby using the combination of the surfactants.

EXAMPLE 8

First reagent (pH 7.0) MOPS (Nacalai Tesque, Inc.) 10 mM HDAOS (DojinKagaku) 0.3 mg/mL Magnesium chloride hexahydrate (Kanto Chemical Co.,Ltd.) 7 mg/dL Sodium dextran sulfate (Tokyo Kasei) 0.7 mg/dL Secondreagent (pH 6.75) MOPS (Nacalai Tesque, Inc.) 50 mM Cholesterol esterase(lipoprotein lipase, 1 U/mL Toyobo Co., Ltd.) Cholesterol oxidase (KyowaHakko 3 U/mL Kogyo Co., Ltd.) Peroxidase (Toyobo Co., Ltd.) 20 U/mL4-Aminoantipyrine (Saikyo Kasei) 0.51 mg/mL Calcium chloride (Wako PureChemical 0.1 mg/mL Industries, Ltd.) Surfactant (kind and concentrationgiven in Table 5)

The test was carried out in a manner similar to Example 4 except thatthe surfactants shown in Table 4 were used and the absorbance wasmeasured immediately after the addition of the second reagent and 5minutes after the addition of the second reagent to obtain thedifferences in the absorbance as A_(LDL), A_(HDL), A_(VLDL) and A_(CM),respectively. Based on the differences, the ratios of A_(HDL)/A_(LDL),A_(VLDL)/A_(LDL) and A_(CM)/A_(LDL) were calculated. The concentrationof cholesterol in each lipoprotein used in this test was determinedusing Determiner L TC II (Kyowa Medex Co., Ltd.) and was found to be 79mg/dL for HDL, 273 mg/dL for LDL, 76 mg/dL for VLDL and 16 mg/dL for CM.

The results are shown in Table 5.

TABLE 5 Concen- Surfactant tration (%) A_(HDL)/A_(LDL) A_(VLDL)/A_(LDL)A_(CM)/A_(LDL) Pluronic L-121 0.4 2.6 4.6 1.3 Emulgen L-40 0.32

As shown in Table 5, LDL cholesterol can be determined more specificallyby using the combination of the surfactants.

EXAMPLE 9

First reagent (pH 7.0) MOPS (Nacalai Tesque, Inc.) 10 mM HDAOS (DojinKagaku) 0.3 mg/mL Magnesium chloride hexahydrate 7 mg/dL (Kanto ChemicalCo., Ltd.) Sodium dextran sulfate (Tokyo Kasei) 0.7 mg/dL Second reagent(pH 7.0) MOPS (Nacalai Tesque, Inc.) 50 mM Cholesterol esterase(lipoprotein lipase, 1 U/mL Toyobo Co., Ltd.) Cholesterol oxidase (KyowaHakko 0.6 U/mL Kogyo Co., Ltd.) Peroxidase (Toyobo Co., Ltd.) 20 U/mL4-Aminoantipyrine (Saikyo Kasei) 0.51 mg/mL Calcium chloride (Wako PureChemical Industries Ltd.) 0.1 mg/mL Surfactant (kind and concentrationgiven in Table 6)

The test was carried out in a manner similar to Example 8 except forusing the surfactants shown in Table 6. A_(LDL), A_(HDL), A_(VLDL) andA_(CM) were determined, respectively, based on which the ratios ofA_(HDL)/A_(LDL), A_(VLDL)/A_(LDL) and A_(CM)/A_(LDL) were calculated.The concentration of cholesterol in each lipoprotein used in this testwas determined using Determiner L TC II (Kyowa Medex Co., Ltd.) and wasfound to be 82 mg/dL for HDL, 270 mg/dL for LDL, 73 mg/dL for VLDL and14 mg/dL for CM.

The results are shown in Table 6.

TABLE 6 Concen- Surfactant tration (%) A_(HDL)/A_(LDL) A_(VLDL)/A_(LDL)A_(CM)/A_(LDL) Pluronic L-121 0.375 2.5 4.3 1.4 Emulgen L-40 0.5Pluronic L-121 0.7125 2.5 2.2 1.8 Emulgen L-40 0.57

As shown in Table 6, LDL cholesterol can be determined more specificallyby using the combination of the surfactants.

EXAMPLE 10

First reagent (pH 7.25) PIPES (Nacalai Tesque, Inc.) 50 mM HDAOS (DojinKagaku) 0.3 mg/mL Second reagent (pH 7.25) PIPES (Nacalai Tesque, Inc.)50 mM Cholesterol esterase (lipoprotein lipase, 2 U/mL Toyobo Co., Ltd.)Cholesterol oxidase (Kyowa Hakko 3 U/mL Kogyo Co., Ltd.) Peroxidase(Toyobo Co., Ltd.) 20 U/,L 4-Aminoantipyrine (Saikyo Kasei) 0.51 mg/mLCalcium chloride (Wako Pure Chemical 0.1 mg/mL Industries, Ltd.) EmulgenL-40 (Kao Corporation) 0.16% Pluronic L-121 (Asahi Denka Kogyo K. K.)0.2%

As human serum samples, 88 samples were collected from the patients andprovided for the quantitative determination of LDL cholesterol in thesamples according to the following procedures.

After 4 μL of each sample was mixed with 300 μL of the first reagent,the mixture was kept at 37° C. for 5 minutes. At this stage, anabsorbance of the mixture was measured. Thereafter, 100 μL of the secondreagent was added to the mixture and reacted. After 5 minutes, anabsorbance of the reaction mixture was measured. Separately, sera withknown concentrations of LDL cholesterol were treated, respectively, inthe same manner. By measuring an absorbance, the concentration ofcholesterol in each sample was quantitatively determined. The absorbancewas measured at a principal wavelength of 600 nm and a secondarywavelength of 700 nm, using Hitachi 7070 autoanalyzer.

On the other hand, total cholesterol, HDL cholesterol and neutral fatwere measured using Determiner L TC (Kyowa Medex Co., Ltd.), DeterminerL HDL-C (Kyowa Medex Co., Ltd.) and Determiner L TG (Kyowa Medex Co.,Ltd.), respectively, which are all commercially available kits. Then theconcentration of LDL cholesterol was determined in accordance with thefollowing Friedewald formula. A correlation coefficient between theconcentration of LDL cholesterol obtained by the method of the presentinvention and the concentration of LDL cholesterol calculated accordingto the Friedewald formula was found to be 0.9767.

Friedewald formula:

(concentration of LDL cholesterol) =(concentration of totalcholesterol)−(concentration of HDL cholesterol)−(concentration ofneutral fat)

FIG. 5 shows the correlation between the concentration of LDLcholesterol obtained by the method of the invention (designated byMethod of the invention in the figure) and the concentration of LDLcholesterol obtained by the comparative method (designated byComparative method in the figure).

INDUSTRIAL APPLICABILITY

The present invention provides the method for the quantitativedetermination of LDL cholesterol and the reagent kit for use in themethod. The present invention also provides the method for continuousfractional determination of HDL cholesterol and LDL cholesterol or totalcholesterol in the same sample in the same system, as well as a reagentkit for use therein.

What is claimed is:
 1. A method for quantitatively determining LDLcholesterol in a biological sample, which comprises: (I) reactingcholesterol in the presence of: a) a biological sample, b) CH enzymesselected from the group consisting of (i) a combination of cholesterolesterase and cholesterol oxidase and (ii) a combination of cholesterolesterase, cholesterol dehydrogenase and oxidized coenzyme, and c) apolyoxyethylene derivative and a polyoxyethylene-polyoxypropylenecopolymer which enable the CH enzymes to act only on LDL cholesterol toform hydrogen peroxide or reduced coenzyme; and (II) measuring theamount of the hydrogen peroxide or reduced coenzyme.
 2. The methodaccording to claim 1, wherein the polyoxyethylene derivative is apolyoxyethylene alkyl ether or a polyoxyethylene alkylaryl ether.
 3. Themethod according to claim 1, wherein thepolyoxyethylene-polyoxypropylene copolymer is a surfactant representedby formula (I): HO—(C₂H₄O)_(a)—(C₃H₆O)_(b)—(C₂H₄O)_(c—H)  (I) wherein a,b and c independently represent an integer of 1 to
 200. 4. The methodaccording to claim 1, wherein the polyoxyethylene derivative is apolyoxyethylene alkyl ether or a polyoxyethylene alkylaryl ether, andthe polyoxyethylene-polyoxypropylene copolymer is a surfactantrepresented by formula (I): HO—(C₂H₄O)_(a—(C) ₃H₆O)_(b)—(C_(2H)₄O)_(c)—H  (I) wherein a, b and c independently represent an integer of1 to
 200. 5. A method for continuous fractional determination of HDLcholesterol and LDL cholesterol in a biological sample, which comprises:(I) subjecting cholesterol to reaction in the presence of: a) abiological sample, b) CH enzymes selected from the group consisting of(i) a combination of cholesterol esterase and cholesterol oxidase and(ii) a combination of cholesterol esterase, cholesterol dehydrogenaseand oxidized coenzyme, and c) a reagent enabling the CH enzyme to actonly on HDL cholesterol to form hydrogen peroxide or reduced coenzyme,(II) measuring an amount of hydrogen peroxide or reduced coenzyme toquantitatively determine the concentration of HDL cholesterol, thenadding a polyoxyethylene derivative and apolyoxyethylene-polyoxypropylene copolymer which enable the CH enzymesto act only on LDL cholesterol; (III) subjecting cholesterol to thereaction to form hydrogen peroxide or reduced coenzyme; (IV) measuringthe amount of the hydrogen peroxide or reduced coenzyme toquantitatively determine the concentration of LDL cholesterol.
 6. Amethod for continuous fractional determination of HDL cholesterol andLDL cholesterol in a biological sample, which comprises: (I) conductinga first reaction of cholesterol in the presence of: a) a biologicalsample, b) CH enzymes selected from the group consisting of (i) acombination of cholesterol esterase and cholesterol oxidase and (ii) acombination of cholesterol esterase, cholesterol dehydrogenase andoxidized coenzyme, and c) a reagent enabling the CH enzymes to act onlyon HDL cholesterol to form hydrogen peroxide or reduced coenzyme, and(II) measuring an amount of hydrogen peroxide or reduced coenzyme toquantitatively determine the concentration of HDL cholesterol, thenadding CH enzymes, and a polyoxyethylene derivative and apolyoxyethylene-polyoxypropylene copolymer which enable the CH enzymesto act only on LDL cholesterol, (III) conducting a second reaction ofcholesterol to form hydrogen peroxide or reduced coenzyme, and measuringthe amount of the hydrogen peroxide or reduced coenzyme toquantitatively determine the concentration of LDL cholesterol.
 7. Themethod according to claim 5 or 6, wherein the polyoxyethylene derivativeis a polyoxyethylene alkyl ether or a polyoxyethylene alkylaryl ether.8. The method according to claim 5 or 6, wherein thepolyoxyethylene-polyoxypropylene copolymer is a surfactant representedby formula (I): HO—(C₂H₄O)_(a)—(C₃H₆O)_(b)—(C₂H₄O)_(c)—H  (I) wherein a,b and c, independently represent an integer of 1 to
 200. 9. The methodaccording to claim 5 or 6, wherein the reagent enabling CH enzyme to actonly on HDL cholesterol is a reagent for aggregating lipoproteins otherthan HDL.
 10. The method according to claim 9, wherein the reagent foraggregating lipoproteins other than HDL is a reagent comprising adivalent metal salt and at least one member selected from the groupconsisting of heparin or a salt thereof, phosphotungstic acid or a saltthereof, dextran sulfuric acid or a salt thereof, polyethylene glycol,sulfated cyclodextrin or a salt thereof, and sulfated oligosaccharide ora salt thereof.
 11. The method according to claim 9, wherein the reagentfor aggregating lipoproteins other than HDL further contains a nonionicsurfactant that does not solubilize the aggregated lipoproteins.
 12. Themethod according to claim 1, 5 or 6, wherein the CH enzymes arecholesterol esterase and cholesterol oxidase, and the determination ofhydrogen peroxide is carried out by reacting the hydrogen peroxide withchromogen in the presence of peroxidase to form a dye and measuring theabsorbance of the reaction mixture.
 13. The method according to claim 5or 6, wherein the polyoxyethylene derivative is a polyoxyethylene alkylether or a polyoxyethylene alkylaryl ether, and thepolyoxyethylene-polyoxypropylene copolymer is a surfactant representedby formula (I): HO—(C₂H₄O)_(a)—(C₃H₆O)_(b)—(C₂H₄O)_(c)—H  (I) wherein a,b and c independently represent an integer of 1 to
 200. 14. The methodaccording to claim 5 or 6, wherein the polyoxyethylene derivative is apolyoxyethylene alkyl ether or a polyoxyethylene alkylaryl ether; thepolyoxyethylene-polyoxypropylene copolymer is a surfactant representedby formula (I): HO—(C₂H₄O)_(a)—(C₃H₆O)_(b)—(C₂H₄O)_(c)—H  (I) wherein a,b and c independently represent an integer of 1 to 200; and the reagentenabling CH enzymes to act only on HDL comprises a (i) divalent metalsalt and (ii) at least one member selected from the group consisting ofheparin or a salt thereof, phosphotungstic acid or a salt thereof,dextran sulfuric acid or a salt thereof, polyethylene glycol, sulfatedcyclodextrin or a salt thereof, and sulfated oligosaccharide ox a saltthereof.
 15. The method according to claim 6, wherein the CH enzymesused in the first reaction are chemically modified enzymes and the CHenzymes used in the second reaction are enzymes that are not chemicallymodified.
 16. A reagent for determining LDL cholesterol comprising CHenzymes selected from the group consisting of (i) a combination ofcholesterol esterase and cholesterol oxidase and (ii) a combination ofcholesterol esterase, cholesterol dehydrogenase and oxidized coenzyme,and a polyoxyethylene derivative and a polyoxyethylene-polyoxypropylenecopolymer which enable the CH enzymes to act only on LDL cholesterol.17. The reagent according to claim 16, wherein the polyoxyethylenederivative is a polyoxyethylene alkylaryl ether.
 18. The reagentaccording to claim 16, wherein the polyoxyethylene-polyoxypropylenecopolymer is a surfactant represented by formula (I):HO—(C₂H₄O)_(a)—(C₃H₆O)_(b)—(C₂H₄O)_(c)—H  (I) wherein a, b and cindependently represent an integer of 1 to
 200. 19. The reagentaccording to claim 16, wherein the CH enzymes are cholesterol esteraseand cholesterol oxidase, and the reagent further comprises peroxidaseand chromogen which produces a dye by reaction with hydrogen peroxide inthe presence of peroxidase.
 20. The reagent according to claim 16,wherein the polyoxyethylene derivative is a polyoxyethylene alkylarylether, and the polyoxyethylene-polyoxypropylene copolymer is asurfactant represented by formula (I):HO—(C₂H₄O)_(a)—(C₃H₆O)_(b)—(C₂H₄O)_(c)—H  (I) wherein a, b and cindependently represent an integer of 1 to
 200. 21. A reagent kit forcontinuous fractional determination of HDL cholesterol and LDLcholesterol comprising a first reagent comprising CH enzymes selectedfrom the group consisting of (i) a combination of cholesterol esteraseand cholesterol oxidase and (ii) a combination of cholesterol esterase,cholesterol dehydrogenase and oxidized coenzyme, and a reagent foraggregating lipoproteins other than HDL, and a second reagent comprisinga polyoxyethylene derivative and a polyoxyethylene-polyoxypropylenecopolymer which enable CH enzymes to act only on LDL cholesterol. 22.The reagent kit according to claim 21, wherein the polyoxyethylenederivative is a polyoxyethylene alkylaryl ether.
 23. The reagent kitaccording to claim 21, wherein the polyoxyethylene-polyoxypropylenecopolymer is a surfactant represented by formula (I):HO—(C₂H₄O)_(a)—(C₃H₆O)_(b)—(C₂H₄O)_(c)—H  (I) wherein a, b and cindependently represent an integer of 1 to
 200. 24. The reagent kitaccording to claim 21, wherein the reagent for aggregating lipoproteinother than HDL is a reagent comprising a divalent metal salt and atleast one member selected from the group consisting of heparin or a saltthereof, phosphotungstic acid or a salt thereof, dextran sulfuric acidor a salt thereof, polyethylene glycol, sulfated cyclodextrin or a saltthereof, and sulfated oligosaccharide or a salt thereof.
 25. The reagentkit according to claim 21, wherein the second reagent further comprisesCH enzymes.
 26. The reagent kit according to claim 25, wherein the CHenzymes in the first reagent are chemically modified enzymes and the CHenzymes in the second reagent are enzymes that are not chemicallymodified.
 27. The reagent kit according to claim 21, wherein the CHenzymes are cholesterol esterase and cholesterol oxidase, and the firstreagent farther comprises peroxidase and chromogen which produces a dyeby reaction with hydrogen peroxide in the presence of peroxidase. 28.The reagent kit according to claim 27, wherein the CH enzymes in thefirst reagent are chemically modified enzymes and the CH enzymes in thesecond reagent are enzymes that are not chemically modified.
 29. Thereagent kit according to claim 21, wherein the reagent for aggregatinglipoproteins other than HDL further contains a nonionic surfactant thatdoes not solubilize the aggregated lipoproteins.
 30. The reagent kitaccording to claim 21, wherein the polyoxyethylene derivative is apolyoxyethylene alkylaryl ether, and thepolyoxyethylene-polyoxypropylene copolymer is a surfactant representedby formula (I): HO—(C₂H₄O)_(a)—(C₃H₆O)_(b)—(C₂H₄O)_(c)—H  (I) wherein a,b and c independently represent an integer of 1 to
 200. 31. The reagentkit according to claim 21, wherein the polyoxyethylene derivative is apolyoxyethylene alkylaryl ether; the polyoxyethylene-polyoxypropylenecopolymer is a surfactant represented by formula (I):HO—(C₂H₄O)_(a)—(C₃H₆O)_(b)—(C₂H₄O)_(c—H)  (I) wherein a, b and cindependently represent an integer of 1 to 200; and the reagent foraggregating lipoproteins other than HDL comprises (i) a divalent metalsalt and (ii) at least one member selected from the group consisting ofheparin or a salt thereof, phosphotungstic acid or a salt thereof,dextran sulfuric acid or a salt thereof, polyethylene glycol, sulfatedcyclodextrin or a salt thereof, and sulfated oligosaccharide or a saltthereof.